Glycosaminoglycans are linear polysaccharides consisting of aminohexose and uronic acid, except of keratin sulfate. They form a large part of intracellular matrix of connective tissue, in particular cartilage, ligaments and tendons. Sulfated polysaccharides, e.g. chondroitin sulfate or dermatan sulfate, are also, besides hyaluronic acid, important examples of glycosaminglycanes.
Chondroitin sulfate is a linear, sulfated, and negatively charged glycosaminoglycan composed of recurrent monomer units of N-acetyl-D-galactosamine and D-glucuronic acid attached to each other via β(1→3) and β(1→4) O-glycosidic bonds (the structural formula of chondroitin sulfate see below).
whereR1 is H or Na,R2 is H, —SO2—OH, or —SO2—ONa
Chondroitin sulfate derives from animal connective tissues, where it binds to proteins and thus forms a part of proteoglycans. The sulfation of chondroitin is realized by means of sulfotransferases in various positions and of various kinds. The unique sulfation pattern of particular positions in the polymer chain encodes the specific biological activity of chondroitin sulfate. Chondroitin sulfate is an important building component of cartilage in joints, conferring them the compression resistance and restoring the balance of the joint lubricant composition (Baeurle S. A., Kiselev M. G., Makarova E. S., Nogovitsin E. A. 2009. Polymer 50: 1805). Together with glucosamine, chondroitin sulfate is used as nutritional supplement for treating or prevention of osteoarthritis in humans (e.g. Flextor®, Advance Nutraceutics, Ltd.) or animals (e.g Gelorendog ®, Contipro Pharma, Ltd.). From the pharmaceutical point of view, chondroitin sulfate is considered to be a drug with delayed response for pain control at degenerative diseases of joints (Aubry-Rozier B. 2012. Revue Médicale Suisse 14: 571).
Dermatan sulfate is linear, sulfated, and negatively charged glycosaminoglycan composed of repeating monomer units of N-acetyl-D-galactosamine and L-iduronic acid attached to each other via β(1→3) and β(1→4) O-glycosidic bonds (the structural formula of dermatan sulfate see below).
whereR1 is H or Na,R2 is H, —SO2—OH, or —SO2—ONa
Dermatan sulfate differs from chondroitin sulfate by the presence of L-iduronic acid, which is a C5 epimer of D-glucuronic acid. The inverse configuration of iduronic acid allows a better flexibility of dermatan sulfate chains and ensures their specific interaction of glycosamine-glycoprotein in the surrounding area. These interactions contribute to the regulation of several cell processes, such as migration, proliferation, differentiation, or angiogenesis. The transformation of chondroitin sulfate into dermatan sulfate is provided by means of three enzymes: dermatan sulfate epimerase 1 (DS-epi1), dermatan sulfate epimerase 2 (DS-epi2), and dermatan 4-O-sulfotransferase (D4ST1). The epimerisation reaction of glucuronic acid into iduronic acid, together with the way of the sulfation, is not random but specifically enzymatically controlled, which results in encoding the information concerning the function of the construed glycosaminoglycan (Thelin M., et al. 2013. FEBS Journal 280: 2431).
Carrageenans are a group of linearly sulfated polysaccharides obtained by the extraction of red marine algae. Galactose and its 3,6-anhydroderivative, that are associated to each other via α(1→3) or β(1→4) O-glycosidic bonds, are their basic building units. There are three main types of carrageenan, which differ in their degree of sulfation and water solubility. Kappa-carrageenan has one sulfate per dimer and forms rigid gels in water. Iota-carrageenan comprises two sulfates and forms soft gels, whereas lambda-carrageenan with three sulfates does not exhibit gel forming properties. Carrageenan is an alternative of animal gelatine for vegetarians and vegans. It is used for thickening and stabilization of food products and as an emulsifier in pharmaceutical and textile industry.
Oxidation of Glycosaminoglycans
Thanks to their functional diversity, the polysaccharides can be oxidized in various positions (Cumpstey I., 2013. ISRN Organic Chemistry, 1). In the case of glycosamineglycanes there are three ways of oxidation. In the first one, the primary hydroxyl is oxidized to form a carboxylic acid. The combination of TEMPO/NaClO is used for the oxidation the most often (Jiang B., et al. 2000. Carbohydrate Research 327: 455; Huang L. et al. 2006. Chemistry, 12: 5264). Due to the steric bulkiness of TEMPO, this method is regioselective for primary hydroxyls only.
On the contrary, the second way leads to the oxidation of secondary hydroxyls to form diketone compounds. In this case, as the oxidation agents the oxides of transition metals based on Cr(VI) (Hassan R., et al. 2013. Carbohydrate Polymers, 92: 2321) or Mn (VII) (Gobouri A. A., et al. 2013. International Journal of Sciences, 2:1; Zaafarany I. A., et al. 2013. Journal of Materials Science Research, 2: 23) are used.
The third type of oxidation is based on periodate (IO4−) oxidation which also attacks secondary hydroxyl groups but simultaneously the pyranose ring breaks (Dawlee S. et al. 2005. Biomacromolecules, 6: 2040; Liang Y., et al. 2011. Colloids and Surfaces B: Biointerfaces, 82: 1; Xu Y., et al. 2012. Carbohydrate Polymers, 87: 1589). During the oxidation, dialdehyde forms first and then it is further oxidized to dicarboxylic acid.
All afore mentioned ways of the oxidation have several drawbacks. In case of the oxidation with the use of TEMPO/NaClO, the formation of polyuronic acid is favoured instead of the desired C6-aldehyde. The reaction conditions for the aldehyde level need to be optimized, as it was demonstrated in the case of hyaluronic acid (Buffa R., et al., WO2011069475, Šedová P., et al., 2013. Carbohydrate Research, 371: 8). In addition, a higher content of carboxylic groups in the polymer significantly influences the conformation, interaction, and recognizing the polysaccharide with the biological surrounding (Zou X. H., et al. 2009. Acta Biomaterialia, 5: 1588).
Even though a chemoselective course of the reaction can be achieved periodate oxidation, this way is not preferred due to the dramatic decrease of the molecular weight of the polymer and irreversible cleavage of the pyranose ring, which results in the loss of the native character of the polysaccharide.
As regards the use of the oxidation agents derived from the transition metal oxides, the oxidized polysaccharides cannot be used for biomedical applications because of their high toxicity (Normandin L., et al. 2002. Metabolic Brain Disease, 17: 375; Katz S. A., et al. 2006. Journal of Applied Toxicology, 13: 217).
Dehydration Reactions of Oxidized Derivatives of Polysaccharides
The presence of an aldehyde in the polysaccharide structure results in an acid character of the hydrogen atom in the adjacent α-position. This hydrogen becomes easily accessible under basic conditions for elimination reactions to form a carbanion, which is stabilized by the conjugation with the adjacent aldehyde and thus displaces the leaving group in the β-position (way a, Scheme 1). The elimination can proceed also under acid conditions, where the activation of the leaving group occurs first to form a carbanion in the β-position (way b, Scheme 1). In the reaction mixture, the carbanion is neutralized with a free electron pair in the α-position. The third possible way can be performed without the addition of a base or an acid, using a simultaneous elimination of a molecule (way c, Scheme 1).

A targeted dehydration of aldehyde of hyaluronan in the 6th position in the glucosamine ring was described in the patent (Buffa et al.: CZ304512). The authors describe the preparation of α,β-unsaturated aldehyde of hyaluronan and its use in cross-linking reactions. The disclosed synthesis involves the use of sterically voluminous organic bases (e.g. diisopropylamine, trimethylamine), inorganic bases, e.g. Ca(OH)2 in the mixture of water-organic solvent of the type of DMSO, sulfolane in the ratio of 3/1 to 1/2 under higher temperatures of 50-60° C. The dehydration is also performed in solid state by heating the polymer to 50-100° C. for 4-5 days. The authors describe the oxidation and dehydration of hyaluronic acid in two steps and they do not describe the direct dehydration during the oxidation step. This solution has an important drawback of two steps synthesis and the use of inappropriate reaction conditions in the presence of caustic (corrosive) elimination agents, presence of an organic solvent, necessity of an elevated temperature, and a long reaction time. All these parameters cause the synthesis to be more expensive and more complicated from the technological point of view (e.g. the corrosion of production apparatus, difficult purification of the product, higher price of dipolar aprotic solvents such as DMSO, sulfolane, and elimination agents such as Et3N and DIPEA, a high consumption of energy and cooling water, a higher risk of dangerous residues in the product, the product biocompatibility at risk, a higher rate of polymer degradation due to the basic environment and higher temperature). The said drawbacks of the synthesis of α,β-unsaturated aldehyde of HA in CZ304512 are, according to this invention, successfully overcome, as the synthesis proceeds in one pot without the necessity to isolate the intermediate product in the form of a saturated C6-aldehyde, without adding the elimination agent, without adding the organic solvent, under room temperature and with the reaction times in the order of hours.
Cross-Linking Reaction of Oxidized Polysaccharides
The introduction of an aldehyde into the polysaccharide structure allows an additional modification of the polymer chain with the aid of nucleophilic addition. Several patent documents describing the binding of amines to aldehydes are known. A typical exemplary reaction for glycosaminoglycans is the reaction of dialdehyde formed by the oxidation with periodate with various low molecular (amines, hydrazides, alkoxyamines, semicarbazides) or polymeric N-nucleophiles (gelatine, chitosan), or S-nucleophiles (thiols, aminothiols) to prepare biocompatible hydrogels (Dawlee S., et al. 2005. Biomacromolecules, 6: 2040; Weng L., et al. 2008. Journal of Biomedical Materials Research part A, 85: 352, Bergman K., et al.: WO2009/108100, Hilborn J., et al.: WO2010/138074). The cross-linking of aldehyde of hyaluronic acid prepared with the use of Dess-Martin periodinane or with the use of the combination of TEMPO/NaClO with various amines was described in patent documents (Buffa R., et al.: WO2011069474; Buffa R., et al.: WO2011069475). α,β-Unsaturated aldehyde of hyaluronic acid was prepared by the dehydration of C6-aldehyde in N-acetyl-D-glucosamine subunit (Buffa R., et al: CZ304512). In addition to oxidized derivatives of hyaluronic acid, the authors describe also its use in reactions with aliphatic, aromatic amines having an optional content of N, S, or O atoms. However, they are prepared under high temperatures and with the use of corrosive elimination agents, which is considerably unfavourable for maintaining their biological activity due to their possible denaturation and the presence of byproducts. Further, the cross-linking reactions of α,β-unsaturated aldehyde of hyaluronic acid with deacetylated polysaccharides as a multifunctional amino linker are mentioned to illustrate the advantages of the conjugation of the aldehyde from the polysaccharide influencing the rheological properties of the prepared hydrogels. However, the hydrogels prepared in this way do not show satisfactory mechanical properties, especially as far as the hydrogel rigidity is concerned.